1. Field of Invention
This invention relates to photolabile compounds and processes for making and using these compounds.
2. Description of Related Art
Light is an essential tool for studying cells. High photonic fluxes are often required to acquire a distinct signal in fluorescence microcopy, but such high power can also disrupt cells (by heating, singlet oxygen production, etc.), and bleach endo/exogenous chromophores. Photolabile “caged” compounds are inert precursors of bioactive molecules that can be loaded into cells and later released in their active form. Photochemical uncaging of biological signaling molecules typically uses brief bursts of light (near-UV wavelengths for regular, one-photon uncaging, or near-IR light for 2-photon photolysis). This mechanism is highly advantageous in studying the kinetics of important signaling events such as, for example, activation of receptors and ion channels and release of neurotransmitters.
U.S. Pat. No. 6,765,014 describes 7-nitroindoline compounds. In section 24, compound 35 is mentioned
among potential candidates for coupling to an effector molecule. However, it is known that the presence of an amino substituents on the nitroaromatic chromophore creates a very low-lying triplet state of the chromophore, and quenches reactivity (Schuddeboom, W. et al., Dipolar Triplet States of p-Nitroaniline and N-Alkyl Derivatives with One-, Two-, and Three-Fold Symmetry J. Phys. Chem. (1996) 100, 12369). Furthermore, it is known that sterically hindered aromatic amines cannot be acylated, even under the best activating condition (Katritzky et al., N-Acylbenzotriazoles: Neutral Acylating Reagents for the Preparation of Primary, Secondary, and Tertiary Amides Org. Chem. 2000; 65, 8210-8213). This is especially true of such amines that are strongly deactivated by one or two electron withdrawing groups that are in the ortho and/or para positions of the aromatic ring. Therefore, dinitroanilines such as compound 35 require radical changes to their structures to make them useful photochemically and synthetically. Therefore, the compound 35 cannot be coupled to an effector molecule without a loss of a nitro group in the 5th position.
WO04085394A1 to Corrie et al. describes 7-nitroindoline compounds which include a triplet sensitizing group such as substituted or unsubstituted benzophenone group and can be used to cage neurotransmitter effector species.
U.S. Pat. Nos. 5,430,175 and 5,587,509 to Hess et al. describe caged carboxyl compounds and methods of releasing carboxyl compounds in which a 2-alkoxy-5-nitrophenyl photosensitive group blocks a carboxyl function. Preferred compounds are caged neuroactive amino acids (e.g., glutamate and gamma-aminobutyric acid (GABA)) with carboxynitrobenzyl chromophores (CNB) photolyzable by laser pulses at wavelengths above about 350 nm within about 3 microseconds and provide a product quantum yield of greater than about 0.2.
The balance between excitatory and inhibitory signaling in the CNS is fundamental to the incredible complexity required for neuronal computation. Chemical synapses are activated by the release of glutamate (Glu), the major excitatory transmitter. This activation is sculpted in many significant ways by the release of γ-aminobutyric acid (GABA), the major inhibitory transmitter. GABA may be released either onto the post-synaptic cell or directly onto a glutamatergic pre-synaptic cell in order to modulate the effects of glutamate release. In the immature brain, GABA is the main excitatory transmitter. GABA releasing synapses are formed before glutamatergic contacts, in many different animals and types of synapses. At some point during development there is an activity-dependent switching from excitation to inhibition by GABAergic synapses.
What is the reason for continued focus on cage development? A brief survey of this history reveals why there is still compelling need for an improved caged glutamate. The first good caged glutamate was CNB-Glu (Weiboldt, et al., 1994). UV-laser excitation released glutamate quickly and with usable efficiency from this caged compound. However, the unadorned ortho-nitrobenzyl chromophore has a very low 2-photon cross section (value of less than 0.001 GM [1 GM=10−50 cm4s/photon]), and CNB-Glu is some what hydrolytically unstable being a benzyl ester. For this reason, Bhc- and DMCNB-Glu were synthesized (Furuta et al., 1999; Ellis-Davies, 1999). The former has a large 2-photon cross section and is totally stable at pH 7.4, however the rate of glutamate photorelease is far too slowly for focal 2-photon uncaging (instead irradiation produces a “mist” of glutamate). The latter releases glutamate quickly and efficiently enough for single spine stimulation but is too unstable in solution before illumination to be impractical for day-to-day use. MNI-Glu was synthesized to solve this latter problem as the benzamido bond is quite stable (solutions stored at 4° C. for 2 d show no detectable hydrolysis, and showed 1% hydrolysis after 13 d, during this time CDNI-Glu had hydrolyzed 7%), and the chromophore preserves the photochemical efficiency of the DMCNB cage. However, when used for 2-photon photolysis MNI-Glu is applied at high concentrations (5-12 mM) that must be irradiated with powers at the very limit of tolerance for brain tissue (Matsuzaki, et al., 2004). For this reason, an even better caged glutamate is desired. An NI-Glu derivative was made containing a cross-linked benzophenone chromophore (“antenna-NI-Glu”, Papageorgiou, et al., 2004). When UV light (300-308 nm) was used to excite the benzophenone antenna, triplet energy transfer to the MNI cage released glutamate with high efficiency, which dropped considerably (ca. 2.5-fold) at 354 nm. Most microscopes do not have a 300-nm laser (standard Ar—Kr lasers emit at 354-363 nm) or quartz optics required for transmission in this region. In contrast, the inventor was able to uncage CDNI-Glu at 354 nm with very high efficiency (similar to the antenna-NI-Glu at 300 nm).
The first caged GABA was made in 1994 (Gee, et al., 1994), by the same group that synthesized CNB-Glu. CNB-GABA has been used by a few groups to study the activation of GABA receptors (Jayaraman, et al., 1999) and to attempt to map the distribution of these receptors in brain on CA1 neurons (Pettit & Augustine, 2000; Eder, et al., 2001). However, even though laser flash photolysis of CNB-GABA suggested that the neurotransmitter is released with a half-time of about 30 microseconds (Gee, et al., 1994), when used to activate currents in outside-out membrane patches with a 10-90% rise time of about 8 ms (Jayaraman, et al., 1999). This latter value is in stark contrast to the values recorded from stellite cells in cerebellar brain slices of 0.4 ms (Nusser, et al., 1997). Rapid flow of saturating [GABA] onto excised outside-out patch using piezo electric devices have given times in the 0.4-0.9 ms (Mozrzymas, et al., 2002; Jones, et al., 1998). Activation of GABA receptors in situ in neurons in brain slices by UV uncaging of CNB-GABA has given rise-times (Pettit & Augustine 2000) that were even slower than excised patches (Jayaraman, et al., 1999). The size of the uncaging spot may contribute to this poor temporal resolution, or the lack of visually identified receptor clusters (as with AMPA receptors on spine heads). The former problem gives a large volume for release that could activate multiple synapses at different distances and times, whilst the latter problem would require uncaged GABA to diffuse after release to the receptor cluster before generating a current; either way poor temporal resolution could be produced. The new caged GABAs as made in accordance with this disclosure and tested in brain slices give the best results so far when judged in terms of how close uncaging can come to mimicking IPSC (compare FIGS. 5 A & B).
Methoxynitroindoline-caged L-glutamate (Papageorgiou & Corrie; Matsuzaki, et al., 2001) and D-aspartate (Huang, et al., 2005) has proved exceptionally useful for neuroscientists and have supplanted the previous generation of CNB-caged compounds.
However, despite the current developments, new much more photoreactive caged transmitters are desired.
All references cited herein are incorporated herein by reference in their entireties.